Background
The amphibian ovarian oocyte is blocked in first meiotic prophase until a transient rise in gonadotropin stimulates its surrounding follicle cells to release progesterone [1], which binds to the first external loop of the catalytic subunit of the Na//K-ATPase at the oocyte surface to reinitiate the meiotic divisions [2,3]. The oocytes then complete one and one-half meiotic divisions, are released from the ovarian follicle, and become blocked at second meiotic metaphase. Sperm penetration results in the completion of meiosis followed by a period of rapid mitoses characteristic of the developing blastula. Rana pipiens ovaries contain a single population of mature oocytes that respond to gonadotropin, in contrast to the multiple growth stages seen in Xenopus laevis when maintained under laboratory conditions [4].
Our previous studies with Rana pipiens oocytes showed that, in vivo, gonadotropin induces phosphorylation of the yolk protein, phosvitin, and that the release of the metaphase block by fertilization and the subsequent synchronous cell divisions coincide with stepwise phosvitin dephosphorylation [5]. In the present study, we have analyzed changes and turnover in high energy phosphates during the meiotic divisions using 31P NMR and 32PO4 pulse labeling techniques in vitro. Rana pipiens oocytes are excellent experimental material for noninvasive NMR studies of cell division because of their large size and the ease of superfusion in an NMR tube, which maintains physiological oxygen levels. Oocytes from each female undergo synchronous meiotic divisions. The prophase-arrested (control) oocytes maintain a sizeable pool of high energy phosphate compounds, including phosphocreatine (PCr), ATP and serine-rich phosphoproteins, for at least 24 h during superfusion [6].
Little has been published about the compartmentation or turnover of high energy phosphates within oocytes or the bioenergetic changes during meiotic division. Using 31P-NMR and the saturation transfer technique, we have examined the effect of the physiological meiotic inducer (progesterone) on both the forward PCr → ATP and the reverse ATP → PCr rates of the creatine kinase reaction in Rana oocytes during the first meiotic division. The 31P-NMR measurements were correlated with in vitro 32PO4 turnover using pulse labeling techniques in oocytes (free of epithelial cells) undergoing synchronous meiotic divisions.
We find that in vitro progesterone initiates an increase in phosphoryl potential and that phosphorylation of the yolk protein phosvitin is accompanied by internalization of the ouabain-sensitive Na/K-ATPase and plasma membrane depolarization. This indicates that a progesterone-induced shift in high energy phosphate utilization from cation pump to phosvitin phosphorylation is necessary for completing the meiotic divisions and early development.